This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding flavanone-3-hydroxylase in plants and seeds.
Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Flavanone-3-hydroxylase, also known as Naringenin, 2-Oxoglutarate 3-Dioxygenase (EC 1.14.11.9), is an enzyme dependent on Fe+2, molecular oxygen, 2-oxoglutarate, and ascorbate, the typical cofactors of the class 2-oxoglutarate-dependent dioxygenases. Flavanone-3-hydroxylase catalyses the 3-beta-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins in plants.
Few of the genes encoding enzymes that regulate these pathways in plants, especially soybeans and Impatiens balsamina, have been isolated and sequenced. cDNAs encoding flavanone-3-hydroxylase have been isolated from petunia, M. incana, carnation, china aster and barley. Residues conserved among similar enzymes have been determined by sequence analysis of various related non-heme iron enzymes. Fourteen amino acids are strictly conserved of which three His and one Asp residues (positions 78, 220, 278 and 222 of the petunia flavanone-3-hydroxylase) have been determined to belong to the putative iron-binding site and an Arg (288) to be part of the 2-oxoglutarate binding site (Britsch L et al. (1993) Eur J Biochem 217:745-754; Lukacin R and Britsch L (1997) Eur J Biochem 249:748-757).
No soybean or Impatiens balsamina genes have yet been reported for flavanone-3-hydroxylase. Accordingly, the availability of nucleic acid sequences encoding all or a portion of flavanone-3-hydroxylase would facilitate studies to better understand the cellular control of the flavonol, anthocyanidin, catechin and proanthocyanidin biosynthetic pathways in soybean and Impatiens balsamina, and provide genetic tools for the manipulation of these pathways.
The instant invention relates to isolated nucleic acid fragments encoding flavanone-3-hydroxylase from soybean and Impatiens balsamina. Specifically, this invention concerns an isolated nucleic acid fragment encoding a flavanone-3-hydroxylase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding flavanone-3-hydroxylase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a flavanone-3-hydroxylase.
In another embodiment, the instant invention relates to a chimeric gene encoding a flavanone-3-hydroxylase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a flavanone-3-hydroxylase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a flavanone-3-hydroxylase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a flavanone-3-hydroxylase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a flavanone-3-hydroxylase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of flavanone-3-hydroxylase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a flavanone-3-hydroxylase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a flavanone-3-hydroxylase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a flavanone-3-hydroxylase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of flavanone-3-hydroxylase in the transformed host cell; (c) optionally purifying the flavanone-3-hydroxylase expressed by the transformed host cell; (d) treating the flavanone-3-hydroxylase with a compound to be tested; and (e) comparing the activity of the flavanone-3-hydroxylase that has been treated with a test compound to the activity of an untreated flavanone-3-hydroxylase, thereby selecting compounds with potential for inhibitory activity.